Polypeptide instability during storage or production of pharmaceutical formulations as well as during purification processes is a well-known problem. Chemical instability in form of formation of soluble aggregates of the polypeptide (covalent dimers and polymers), deamidation, oxidation and isomerisation etc. are well known problems difficult to avoid due to the relatively labile nature of most polypeptides. Chemical instability of polypeptides in pharmaceutical formulations therefore causes significant problems for production and storage of polypeptide pharmaceuticals and methods for reduction or elimination of this problem are of considerable interest to the pharmaceutical industry. Formation of such chemical degradation products is often accelerated with increasing temperature; hence restrictions regarding exposure to elevated temperatures (i.e. above for instance 8° C.) during distribution, storage and use are often necessary to ensure sufficient quality of the pharmaceutical polypeptide. As a consequence, the patient must often store the protein/peptide pharmaceutical in the fridge to depress the inevitable accelerated chemical degradation at room temperature.
Among the various chemical degradation pathways of pharmaceutical polypeptides, special attention is often paid to aggregation due to the reduced biological potency and potential antigenic properties associated with such aggregated forms. Some aggregates involve formation of new covalent bond(s) for instance through disulfide-bond formation between free Cysteine residues, transamidation, dityrosine formation or formaldehyde-mediated crosslinking (see also Wang, W. Int. J. Pharm. 289:1-30, 2005). Transamidation concerns reaction between free amino groups (eg. N-terminals) from one polypeptide with amide groups (Asparagine or Glutamine residues) of another polypeptide. Formation of transamidation dimers and polymers in insulin formulation have been reported by Brange and co-workers (Brange, J. Pharm Res 9:727-734, 1992).
In pharmaceutical formulations of insulin, reduction of the formation of covalent dimers and polymers are of particular interest due to their markedly reduced biological potency compared to the native insulin molecule and to their potential antigenic properties. Consequently, insulin formulations stabilized against chemical degradation, in particular formation of covalent dimers and polymers, are highly desirable.
The addition of buffers to polypeptide-containing pharmaceutical solutions is essential in order to stabilize pH of the purification or pharmaceutical solution within a desired pH range. Conventionally, phosphate buffers have been used as the preferred buffering agent for pharmaceutical formulations containing polypeptides.
US20030125234 discloses alteration of protein physical stability (formation of insoluble aggregates or fibrils) by multiple charged compounds. Ethylenediamine and insulin are presented, however, no significant positive effect on physical stability of insulin is observed in the presence of ethylenediamine. Chemical stability (e.g. formation of soluble aggregates, e.g. covalent dimer and polymer), which is the topic of the present invention, is not evaluated.